Project Summary/Abstract Telomerase Reverse Transcriptase (TERT) is the primary enzyme critical for maintaining telomere length, but is normally silenced in somatic cells to limit the amount of times a cell can replicate. In order for somatic cells to become tumorigenic, they must re-activate the telomerase complex by re-enabling TERT expression. The most common way that many cancers re-activate telomerase is by acquiring an activating mutation in the promoter region of the TERT gene. These TERT promoter mutations occur in over 50 types of cancer, including over 80% of primary glioblastomas (GBM), and place TERT expression under the control of the GABP transcription factor complex in tumors that bear them. I have previously discovered that a specific GABP transcription factor complex, the GABPB1L-containing GABP tetramer, is recruited to the mutant TERT promoter and is required for telomerase reactivation in GBM. I found that disruption of this GABP tetramer complex via CRISPR/Cas9 editing was sufficient to reverse cellular immortality exclusively in GBM lines bearing TERT promoter mutations. Strikingly, the TERT promoter wild-type lines lacking GABPB1L displayed no phenotype, suggesting that this GABP tetramer complex is dispensable for normal cell function. Furthermore, further analysis revealed that TERT promoter mutant lines lacking GABPB1L were dying from the high- level genomic instability, a previously described mechanism of cell death with tangible therapeutic implications. These initial findings have raised two main questions the F99 research will address: (1) Why is the GABPB1L- containing GABP tetramer dispensable for normal cell function, but GABPB1L deficiency is lethal to TERT promoter mutant cancers? Understanding the specificity of GABPB1L for the mutant TERT promoter is key for assessing its candidacy as a new therapeutic target in TERT promoter mutant GBM. Answering this question will require the use of high-throughput sequencing and bioinformatics analysis, skills employed by the Costello lab. (2) Can we combine standard therapies for GBM with the ablation of GABPB1L to achieve synergistic anti-tumor effects? Genomic instability induced by TERT depletion sensitizes cancer cells to DNA damaging agents. Thus, I propose to evaluate the potential for synergy between GABPB1L inhibition, subsequent TERT depletion and current GBM therapies in TERT promoter-mutant GBM. I expect that my efforts will provide insight into this potential therapy targeting GBM immortality while also providing me with training critical to my career as a researcher in cancer cell immortality. For the K00 phase, I propose to leverage the broad range of conceptual and technical expertise I have amassed studying TERT promoter mutations on a mechanistic level in GBM to complete a postdoctoral fellowship studying cellular immortality as a therapeutic target in cancer. Although I hope to continue to study TERT promoter mutations, I plan to expand my knowledge base by studying other mechanisms of cancer immortality in GBM and cancer types such as lung or colon cancer. Specifically, I will combine my previous research experience with advanced training in in vivo models of cancer, bioinformatics, cancer therapeutics, and molecular biology to provide new insight into the mechanisms governing cancer cell immortality and to develop novel therapeutic strategies targeting these mechanisms.